Cystic Fibrosis and Point Mutation Detection using MutS

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Cystic Fibrosis and Point Mutation Detection
using MutS

• Marilyn Wadzanai Shumba
• Biology Department
• Susquehanna University
• 514 University Avenue
• Selinsgrove PA 17870

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Part I
Introduction

• Cystic Fibrosis

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Cystic Fibrosis

• a hereditary human disorder characterized by
  chronic pulmonary infection
• the most common type of chronic lung disease in
  youth
• causes accumulation of thick, sticky mucus in the
  lungs and digestive tract
• life-threatening lung infections and serious
  digestion problems

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Cystic Fibrosis continued

• Mucus that accumulates in the the pancrease make
  it hard to break down and absorb food.
• CF may also affect the sweat glands and a man's
  reproductive system.

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CF is recessive inheritance disorder
Inheritance
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Inheritance continued

• millions of Americans are carriers
• common among Europeans of Northern or Central
  descent
• estimated 3 to 10 of Caucasians have the CF
  gene
• most common, deadly, inherited disorder in the
  United States

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Discovery of CF gene

• Linkage Analysis
• Collect samples from families affected by disease
• Samples examined for the presence of physical
  markers such as single nucleotide polymorphisms
  (SNPs)
• CF close to SNPs

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Part II
Molecular Biology

• Mutation Detection in CF using Muts

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CF Gene mutations

• Cystic Fibrosis Transmembrane Conductance
  Regulator (CFTR) protein mediates Cl- ion flow
  across membrane
• The deletion of the codon for phenylalanine at
  position 508 (?F508) produces a defective CFTR
• Disrupts ion flow

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CF Gene mutations continued

• Over 170 different CF mutations exist
• Approx. 70 of CF carriers have the gene mutant
  allele, ?F508

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MutS

• DNA repair enzymes
• Binds DNA mismatches
• Distinguish between heterozygotes and homozygotes
  in genomic
• Screening genomes for polymorphisms and disease
  causing mutations

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Detection of CF carriers

• Muts binds strongly to heteroduplex DNA that
  corresponds to heterozygous carriers (of ?F508)
• Deletion of ?F508 generates kinked heteroduplexes
• Reduced electrophoretic mobility

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Detection of CF carries continued

• Exploiting conformational differences of
  mismatched heteroduplexes using
• Denaturing gradient gel electrophoresis
• Single stranded conformational polymorphisms
  revealed by gel electrophoresis

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Advantages of using Muts

• Small amounts of DNA required for examination
• Simple and fact staining
• No demand for PCR product purification
• No labelling and radioisotopes required
• Muts proteins binds different mismatches with
  different affinities

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Disadvantages of using Muts

• Not easiest method for detecting heterozygosity
  for ?F508
• MutS binds both heteroduplexes and homoduplexes
• It cannot distinguish the homozygotes with both
  wild type or mutant alleles
• Detection of point mutations using
  single-stranded polymorphism, denaturing gradient
  gel electrophoresis, or mutation detection
  enhancement gels require long or complicated
  electrophoretic runs

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Significance

• MutS used to detect point mutations
• Carriers can be identified
• No prevention
• ?Family planning
• ?Early detection

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Early detection Screening for CF

• Routine newborn (neonatal) screening for CF
• blood sample taken six days after birth to test
  for low thyroid function and phenylketonuria.
• Chorionic villus sampling
• Taking a biopsy from the placenta and analysing
  the DNA

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Later Tests

• first blood tests for CF approved in May 2005 FDA
  
• look for variations in a CF gene
• Sweat chloride test.
• Fecal fat test
• Upper GI and small bowel series
• Measurement of pancreatic function

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Carrier Assays for CF

• Techniques
• DNA amplification
• Restriction enzyme digestion
• Gel electrophoresis and
• Southern transfer, dot-blotting, and probe
  hybridization

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Symptoms

• Because there are more than 1,000 mutations of
  the CF gene, symptoms differ from person to
  person

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Treatment

• Early diagnosis
• Antibiotics for respiratory infections.
• Replacement of Pancreatic enzymes, or DNAse
  enzyme replacement therapy
• Vitamins A, D, E, and K.
• Inhaled bronchodilators albuterol (Proventil,
  Ventolin), help open the airways.
• Pain relievers to slow lung deterioration in
  minors
• Postural drainage and chest percussion.
• Lung transplant

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References

• Alberts, Johnson, Lewis, Raff, Roberts, Walter
  Molecular Biology of the Cell, 4th Ed. Garland
  Science NY 2002
• Lishanski et al.
• Stanislawska-Sachadyn Preliminary studies on DNA
  retardation by MutS applied to the detection of
  point mutations in clinical samples Science
  Direct 2005 Feb 15 lthttp//sciencedirect.comgt
• BupaCystic Fibrosis2006
• lthttp//hcd2.bupa.co.uk/fact_sheets/Mosby_factshe
  ets/Cystic_fibrosis.htmlgt
• Owens, Thomas Cystic Fibrosis Medline Plus
  2006 lthttp//www.nlm.nih.gov/medlineplus/ency/arti
  cle/000107.htmgt
• U.S Congress, Office of Technology Assessment,
  Cystic Fibrosis and DNA Tests Implications of
  Carrier Screening, OTA-BA-532 (Washington, DC
  U.S. Government Printing Office, August
  1992)lthttp//www.wws.princeton.edu/ota/disk1/1992
  /9208/9208.PDFgt